The bio-electronic interface is a key component in biological research and biomedical applications. Existing techniques face many intrinsic challenges due to the size and mechanical mismatch between recording electrodes and live cells, which leads to limitations in their performance, biocompatibility and lifetime, especially fr implanted applications. The fundamental question is: can we re-examine the basic unit of such interface to develop bio-probes from bottom-up, so that the artificial electronics and the biological network can be bridged in a more natural way? Nanowire-based field-effect transistor (FET) sensors have shown high sensitivity in detecting biological signals as well as rich interactions with live cells, owing to their nanoscale size and structure. This makes them very promising building blocks for constructing high-performance, minimally-invasive bio-probes for in vitro and in vivo applications. The overall goal of this proposed R21 project is to develop ultra-small free-standing nanowire transistor bio-probes for multiplexed intracellular study and implanted biomedical applications. In our on-going experiments, we discovered that a nanoscale field-effect transistor (nanoFET) can be synthetically integrated in a kinked Si nanowire (SiNW) to build a three-dimensional (3D) nano-sensor, which can non-invasively enter live cardiomyocytes to obtain full intracellular action potentials. We hypothesize that this method can be developed into a general platform for interfacing with different types of cells, and more importantly, for building implantable bio-probes with unique interplay with live cells, by incorporating biodegradable sacrificial layers that allow post-implantation in situ formation of flexible 3D structures to promote tighter interactions with active cells away from the more rigid supportive body. This overall hypothesis will be addressed in the experiments of the following Specific Aims: (1) to develop general protocols for promoting strong nanoFET-cell interaction and (2) to develop ultra-small free- standing nanoFET probes optimized for intracellular recording and implanted applications. The systematic study here would bring insightful understanding and control of the nanowire-cell interaction, and provide robust protocols for using nanowire-based probes for physiological study and biomedical applications. In addition, the unique design of free-standing probe with three-dimensional (3D) flexible structure formed in situ after implantation, which presents the nanoFET in 3D free space without bulky supporting substrates, could greatly enhance signal quality and reproducibility, and expand the functionality of the nanoelectronic sensors, due to the significantly reduced probe size and better mechanical matching with the tissue, leading to less tissue reaction, and more natural coupling with cells. Our study would find broad biomedical applications for assistive devices, prosthesis and brain-machine interface.